The long term objective of this study is to gain a molecular understanding of epigenetic processes of gene silencing and chromatin remodeling. PEV in Drosophila has served as a major paradigm for the identification and genetic analysis of evolutionary conserved determinants of epigenetic regulation of chromatin structure and gene silencing. We present evidence that the JIL-1 histone H3S10 chromosomal kinase functions to maintain euchromatic regions by antagonizing heterochromatization and gene silencing at ectopic locations by methyltransferase mediated histone H3K9 dimethylation and HP1 recruitment. Consequently, to understand regulation of heterochromatin formation and gene silencing in Drosophila it will be crucial to determine the molecular mechanisms of JIL-1's role in this process and JIL-Ts placement in the genetic hierarchy. Thus, we will test the hypothesis that JIL-1 functions to counterbalance the spread of heterochromatic factors by 1) determining the changes in the distribution of chromatin markers that are diagnostic for active (euchromatic) or silenced (heterochromatic) chromatin in JIL-1 mutant backgrounds, by 2) conducting genetic experiments to define the in vivo pathway in which JIL-1 functions to maintain chromosomal domains and determine JIL-Ts place in the genetic hierarchy, and by 3) determining the molecular mechanisms underlying the loss of viability and perturbation of chromatin structure in JIL-1 mutant backgrounds. Furthermore, we propose that changes in PEV observed in JIL-1 loss-of-function mutants will be accompanied by changes in chromatin structure at the nucleosomal level in a chromatin context-dependent manner. To test these hypotheses we will conduct a series of genetic and biochemical experiments designed to examine how JIL-1 affects nucleosome organization and PEV using chromosomal inversions and insertions of a reporter gene into different chromatin environments. We will directly test the hypothesis that alterations in chromatin structure are caused by the phosphorylation state of histone H3S10 by targeting JIL-1 to ectopic chromosome locations. Finally, we will define the specific domains of JIL-1 that are necessary for chromatin structure regulation in PEV and determine the role of kinase activity in this process by generating "kinase dead" constructs and expressing the mutated constructs of JIL-1 transgenically in null mutant flies. Gene silencing is a critical developmental process relevant to many human health problems that include cancer. Thus, the proposed studies of JIL-1 will provide important new insights into the molecular mechanisms of how kinase activity modulates chromatin structure and gene regulation that are directly relevant to humans. [unreadable] [unreadable] [unreadable]